Overdrive is essential for targeted sperm elimination by Segregation Distorter.

Intra-genomic conflict driven by selfish chromosomes is a powerful force that shapes the evolution of genomes and species. In the male germline, many selfish chromosomes bias transmission in their own favor by eliminating spermatids bearing the competing homologous chromosomes. However, the mechanisms of targeted gamete elimination remain mysterious. Here, we show that Overdrive (Ovd), a gene required for both segregation distortion and male sterility in Drosophila pseudoobscura hybrids, is broadly conserved in Dipteran insects but dispensable for viability and fertility. In D. melanogaster, Ovd is required for targeted Responder spermatid elimination after the histone-to-protamine transition in the classical Segregation Distorter system. We propose that Ovd functions as a general spermatid quality checkpoint that is hijacked by independent selfish chromosomes to eliminate competing gametes.

Decoding chromosome organization using CheC-PLS: chromosome conformation by proximity labeling and long-read sequencing.

Genomic approaches have provided detailed insight into chromosome architecture. However, commonly deployed techniques do not preserve connectivity-based information, leaving large-scale genome organization poorly characterized. Here, we developed CheC-PLS: a proximity-labeling technique that indelibly marks, and then decodes, protein-associated sites. CheC-PLS tethers dam methyltransferase to a protein of interest, followed by Nanopore sequencing to identify methylated bases - indicative of in vivo proximity - along reads >100kb. As proof-of-concept we analyzed, in budding yeast, a cohesin-based meiotic backbone that organizes chromatin into an array of loops. Our data recapitulates previously obtained association patterns, and, importantly, exposes variability between cells. Single read data reveals cohesin translocation on DNA and, by anchoring reads onto unique regions, we define the internal organization of the ribosomal DNA locus. Our versatile technique, which we also deployed on isolated nuclei with nanobodies, promises to illuminate diverse chromosomal processes by describing the in vivo conformations of single chromosomes.

Transformation of meiotic drive into hybrid sterility in Drosophila.

Hybrid male sterility is one of the fastest evolving intrinsic reproductive barriers between recently isolated populations. A leading explanation for the evolution of hybrid male sterility involves genomic conflicts with meiotic drivers in the male germline. There are, however, few examples directly linking meiotic drive to hybrid sterility. Here, we report that the Sex-Ratio chromosome of Drosophila pseudoobscura, which causes X-chromosome drive within the USA subspecies, causes near complete male sterility when moved into the genetic background of the Bogota subspecies. In addition, we show that this new form of sterility is genetically distinct from the sterility of F1 hybrid males in crosses between USA males and Bogota females. Our observations provide a tractable study system where non-cryptic drive within species is transformed into strong hybrid sterility between very young subspecies.

A new hybrid incompatibility locus between Drosophila melanogaster and Drosophila sechellia.

Despite the fundamental importance of hybrid incompatibilities to the process of speciation, there are few cases where the evolution and genetic architecture of hybrid incompatibilities are understood. One of the longest studied hybrid incompatibilities causes F1 hybrid male inviability in crosses between Drosophila melanogaster females and males from the Drosophila simulans clade of species-Drosophila simulans, Drosophila mauritiana, and Drosophila sechellia. Here, we discover dramatic differences in the manifestation of this lethal hybrid incompatibility among the D. simulans clade of species. In particular, F1 hybrid males between D. melanogaster and D. sechellia are resistant to hybrid rescue through RNAi knockdown of an essential hybrid incompatibility gene. To understand the genetic basis of this inter-species difference in hybrid rescue, we developed a triple-hybrid mapping method. Our results show that 2 discrete large effect loci and many dispersed small effect changes across the genome underlie D. sechellia aversion to hybrid rescue. The large effect loci encompass a known incompatibility gene Lethal hybrid rescue (Lhr) and previously unknown factor, Sechellia aversion to hybrid rescue (Satyr). These results show that the genetic architecture of F1 hybrid male inviability is overlapping but not identical in the 3 inter-species crosses. Our results raise questions about whether new hybrid incompatibility genes can integrate into an existing hybrid incompatibility thus increasing in complexity over time, or if the continued evolution of genes can gradually strengthen an existing hybrid incompatibility.

Genome evolution: A story of species and satellites.

Satellite DNA sequences can rapidly expand, and pressure to preserve genome integrity is thought to trigger the adaptive evolution of satellite-associated proteins. The authors of a new study manipulate both sides of this co-evolution in Drosophila to reveal how DNA entanglements can trigger the rapid adaptive evolution of chromatin proteins.

Extensive Recombination Suppression and Epistatic Selection Causes Chromosome-Wide Differentiation of a Selfish Sex Chromosome in Drosophila pseudoobscura.

Sex-Ratio (SR) chromosomes are selfish X-chromosomes that distort Mendelian segregation and are commonly associated with inversions. These chromosomal rearrangements suppress recombination with Standard (ST) X-chromosomes and are hypothesized to maintain multiple alleles important for distortion in a single large haplotype. Here, we conduct a multifaceted study of the multiply inverted Drosophila pseudoobscura SR chromosome to understand the evolutionary history, genetic architecture, and present-day dynamics that shape this enigmatic selfish chromosome. The D. pseudoobscura SR chromosome has three nonoverlapping inversions of the right arm of the metacentric X-chromosome: basal, medial, and terminal. We find that 23 of 29 Mb of the D. pseudoobscuraX-chromosome right arm is highly differentiated between the Standard and SR arrangements, including a 6.6 Mb collinear region between the medial and terminal inversions. Although crossing-over is heavily suppressed on this chromosome arm, we discover it is not completely eliminated, with measured rates indicating recombination suppression alone cannot explain patterns of differentiation or the near-perfect association of the three SR chromosome inversions in nature. We then demonstrate the ancient basal and medial inversions of the SR chromosome contain genes sufficient to cause weak distortion. In contrast, the younger terminal inversion cannot distort by itself, but contains at least one modifier gene necessary for full manifestation of strong sex chromosome distortion. By parameterizing population genetic models for chromosome-wide linkage disequilibrium with our experimental results, we infer that strong selection acts to maintain the near-perfect association of SR chromosome inversions in present-day populations. Based on comparative genomic analyses, direct recombination experiments, segregation distortion assays, and population genetic modeling, we conclude the combined action of suppressed recombination and strong, ongoing, epistatic selection shape the D. pseudoobscura SR arrangement into a highly differentiated chromosome.

Recurrent Losses and Rapid Evolution of the Condensin II Complex in Insects.

Condensins play a crucial role in the organization of genetic material by compacting and disentangling chromosomes. Based on studies in a few model organisms, the condensins I and II complexes are considered to have distinct functions, with the condensin II complex playing a role in meiosis and somatic pairing of homologous chromosomes in Drosophila. Intriguingly, the Cap-G2 subunit of condensin II is absent in Drosophila melanogaster, and this loss may be related to the high levels of chromosome pairing seen in flies. Here, we find that all three non-SMC subunits of condensin II (Cap-G2, Cap-D3, and Cap-H2) have been repeatedly and independently lost in taxa representing multiple insect orders, with some taxa lacking all three. We also find that all non-Dipteran insects display near-uniform low-pairing levels regardless of their condensin II complex composition, suggesting that some key aspects of genome organization are robust to condensin II subunit losses. Finally, we observe consistent signatures of positive selection in condensin subunits across flies and mammals. These findings suggest that these ancient complexes are far more evolutionarily labile than previously suspected, and are at the crossroads of several forms of genomic conflicts. Our results raise fundamental questions about the specific functions of the two condensin complexes in taxa that have experienced subunit losses, and open the door to further investigations to elucidate the diversity of molecular mechanisms that underlie genome organization across various life forms.

Altered Localization of Hybrid Incompatibility Proteins in Drosophila.

Understanding the molecular basis of hybrid incompatibilities is a fundamental pursuit in evolutionary genetics. In crosses between Drosophila melanogaster females and Drosophila simulans males, an interaction between at least three genes is necessary for hybrid male lethality: Hmr mel, Lhr sim, and gfzf sim. Although HMR and LHR physically bind each other and function together in a single complex, the connection between gfzf and either of these proteins remains mysterious. Here, we show that GFZF localizes to many regions of the genome in both D. melanogaster and D. simulans, including at telomeric retrotransposon repeats. We find that GFZF localization at telomeres is significantly different between these two species, reflecting the rapid evolution of telomeric retrotransposon copy number composition between the two species. Next, we show that GFZF and HMR normally do not colocalize in D. melanogaster. In interspecies hybrids, however, HMR shows extensive mis-localization to GFZF sites, thus uncovering a new molecular interaction between these hybrid incompatibility factors. We find that spreading of HMR to GFZF sites requires gfzf sim but not Lhr sim, suggesting distinct roles for these factors in the hybrid incompatibility. Finally, we find that overexpression of HMR and LHR within species is sufficient to mis-localize HMR to GFZF binding sites, indicating that HMR has a natural low affinity for GFZF sites. Together, these studies provide the first insights into the different properties of gfzf between D. melanogaster and D. simulans, and uncover a molecular interaction between gfzf and Hmr in the form of altered protein localization.

How chromosomal rearrangements shape adaptation and speciation: Case studies in Drosophila pseudoobscura and its sibling species Drosophila persimilis.

The gene arrangements of Drosophila have played a prominent role in the history of evolutionary biology from the original quantification of genetic diversity to current studies of the mechanisms for the origin and establishment of new inversion mutations within populations and their subsequent fixation between species supporting reproductive barriers. This review examines the genetic causes and consequences of inversions as recombination suppressors and the role that recombination suppression plays in establishing inversions in populations as they are involved in adaptation within heterogeneous environments. This often results in the formation of clines of gene arrangement frequencies among populations. Recombination suppression leads to the differentiation of the gene arrangements which may accelerate the accumulation of fixed genetic differences among populations. If these fixed mutations cause incompatibilities, then inversions pose important reproductive barriers between species. This review uses the evolution of inversions in Drosophila pseudoobscura and D. persimilis as a case study for how inversions originate, establish and contribute to the evolution of reproductive isolation.

Ancestral polymorphisms explain the role of chromosomal inversions in speciation.

Understanding the role of chromosomal inversions in speciation is a fundamental problem in evolutionary genetics. Here, we perform a comprehensive reconstruction of the evolutionary histories of the chromosomal inversions in Drosophila persimilis and D. pseudoobscura. We provide a solution to the puzzling origins of the selfish Sex-Ratio arrangement in D. persimilis and uncover surprising patterns of phylogenetic discordance on this chromosome. These patterns show that, contrary to widely held views, all fixed chromosomal inversions between D. persimilis and D. pseudoobscura were already present in their ancestral population long before the species split. Our results suggest that patterns of higher genomic divergence and an association of reproductive isolation genes with chromosomal inversions may be a direct consequence of incomplete lineage sorting of ancestral polymorphisms. These findings force a reconsideration of the role of chromosomal inversions in speciation, not as protectors of existing hybrid incompatibilities, but as fertile grounds for their formation.

Parallel Evolution of Sperm Hyper-Activation Ca2+ Channels.

Sperm hyper-activation is a dramatic change in sperm behavior where mature sperm burst into a final sprint in the race to the egg. The mechanism of sperm hyper-activation in many metazoans, including humans, consists of a jolt of Ca2+ into the sperm flagellum via CatSper ion channels. Surprisingly, all nine CatSper genes have been independently lost in several animal lineages. In Drosophila, sperm hyper-activation is performed through the cooption of the polycystic kidney disease 2 (pkd2) Ca2+ channel. The parallels between CatSpers in primates and pkd2 in Drosophila provide a unique opportunity to examine the molecular evolution of the sperm hyper-activation machinery in two independent, nonhomologous calcium channels separated by > 500 million years of divergence. Here, we use a comprehensive phylogenomic approach to investigate the selective pressures on these sperm hyper-activation channels. First, we find that the entire CatSper complex evolves rapidly under recurrent positive selection in primates. Second, we find that pkd2 has parallel patterns of adaptive evolution in Drosophila. Third, we show that this adaptive evolution of pkd2 is driven by its role in sperm hyper-activation. These patterns of selection suggest that the evolution of the sperm hyper-activation machinery is driven by sexual conflict with antagonistic ligands that modulate channel activity. Together, our results add sperm hyper-activation channels to the class of fast evolving reproductive proteins and provide insights into the mechanisms used by the sexes to manipulate sperm behavior.

A genomic approach to identify hybrid incompatibility genes.

Uncovering the genetic and molecular basis of barriers to gene flow between populations is key to understanding how new species are born. Intrinsic postzygotic reproductive barriers such as hybrid sterility and hybrid inviability are caused by deleterious genetic interactions known as hybrid incompatibilities. The difficulty in identifying these hybrid incompatibility genes remains a rate-limiting step in our understanding of the molecular basis of speciation. We recently described how whole genome sequencing can be applied to identify hybrid incompatibility genes, even from genetically terminal hybrids. Using this approach, we discovered a new hybrid incompatibility gene, gfzf, between Drosophila melanogaster and Drosophila simulans, and found that it plays an essential role in cell cycle regulation. Here, we discuss the history of the hunt for incompatibility genes between these species, discuss the molecular roles of gfzf in cell cycle regulation, and explore how intragenomic conflict drives the evolution of fundamental cellular mechanisms that lead to the developmental arrest of hybrids.

An essential cell cycle regulation gene causes hybrid inviability in Drosophila.

Speciation, the process by which new biological species arise, involves the evolution of reproductive barriers, such as hybrid sterility or inviability between populations. However, identifying hybrid incompatibility genes remains a key obstacle in understanding the molecular basis of reproductive isolation. We devised a genomic screen, which identified a cell cycle-regulation gene as the cause of male inviability in hybrids resulting from a cross between Drosophila melanogaster and D. simulans. Ablation of the D. simulans allele of this gene is sufficient to rescue the adult viability of hybrid males. This dominantly acting cell cycle regulator causes mitotic arrest and, thereby, inviability of male hybrid larvae. Our genomic method provides a facile means to accelerate the identification of hybrid incompatibility genes in other model and nonmodel systems.

Drosophila melanogaster activating transcription factor 4 regulates glycolysis during endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress results from an imbalance between the load of proteins entering the secretory pathway and the ability of the ER to fold and process them. The response to ER stress is mediated by a collection of signaling pathways termed the unfolded protein response, which plays important roles in development and disease. Here we show that in Drosophila melanogaster S2 cells, ER stress induces a coordinated change in the expression of genes involved in carbon metabolism. Genes encoding enzymes that carry out glycolysis were up-regulated, whereas genes encoding proteins in the tricarboxylic acid cycle and respiratory chain complexes were down-regulated. The unfolded protein response transcription factor Atf4 was necessary for the up-regulation of glycolytic enzymes and Lactate dehydrogenase (Ldh). Furthermore, Atf4 binding motifs in promoters for these genes could partially account for their regulation during ER stress. Finally, flies up-regulated Ldh and produced more lactate when subjected to ER stress. Together, these results suggest that Atf4 mediates a shift from a metabolism based on oxidative phosphorylation to one more heavily reliant on glycolysis, reminiscent of aerobic glycolysis or the Warburg effect observed in cancer and other proliferative cells.

Speciation via autoimmunity: a dangerous mix.

In this issue of Cell, Chae et al. find that genomic "hot spots" encoding NLR plant immune receptor genes are recurrently responsible for hybrid necrosis, highlighting the role of host-pathogen evolutionary arms races in driving the evolution of hybrid incompatibilities.

Birth, death, and replacement of karyopherins in Drosophila.

Nucleocytoplasmic transport is a broadly conserved process across eukaryotes. Despite its essential function and conserved mechanism, components of the nuclear transport apparatus have been implicated in genetic conflicts in Drosophila, especially in the male germ line. The best understood case is represented by a truncated RanGAP gene duplication that is part of the segregation distorter system in Drosophila melanogaster. Consistent with the hypothesis that the nuclear transport pathway is at the heart of mediating genetic conflicts, both nucleoporins and directionality imposing components of nuclear transport have previously been shown to evolve under positive selection. Here, we present a comprehensive phylogenomic analysis of importins (karyopherins) in Drosophila evolution. Importins are adaptor molecules that physically mediate the transport of cargo molecules and comprise the third component of the nuclear transport apparatus. We find that importins have been repeatedly gained and lost throughout various stages of Drosophila evolution, including two intriguing examples of an apparently coincident loss and gain of nonorthologous and noncanonical importin-α. Although there are a few signatures of episodic positive selection, genetic innovation in importin evolution is more evident in patterns of recurrent gene birth and loss specifically for function in Drosophila testes, which is consistent with their role in supporting host genomes defense against segregation distortion.

Genetic architecture of male sterility and segregation distortion in Drosophila pseudoobscura Bogota-USA hybrids.

Understanding the genetic basis of reproductive isolation between recently diverged species is a central problem in evolutionary genetics. Here, I present analyses of the genetic architecture underlying hybrid male sterility and segregation distortion between the Bogota and USA subspecies of Drosophila pseudoobscura. Previously, a single gene, Overdrive (Ovd), was shown to be necessary but not sufficient for both male sterility and segregation distortion in F(1) hybrids between these subspecies, requiring several interacting partner loci for full manifestation of hybrid phenomena. I map these partner loci separately on the Bogota X chromosome and USA autosomes using a combination of different mapping strategies. I find that hybrid sterility involves a single hybrid incompatibility of at least seven interacting partner genes that includes three large-effect loci. Segregation distortion involves three loci on the Bogota X chromosome and one locus on the autosomes. The genetic bases of hybrid sterility and segregation distortion are at least partially--but not completely--overlapping. My results lay the foundation for fine-mapping experiments to identify the complete set of genes that interact with Overdrive. While individual genes that cause hybrid sterility or inviability have been identified in a few cases, my analysis provides a comprehensive look at the genetic architecture of all components of a hybrid incompatibility underlying F(1) hybrid sterility. Such an analysis would likely be unfeasible for most species pairs due to their divergence time and emphasizes the importance of young species pairs such as the D. pseudoobscura subspecies studied here.

Accelerated evolution of the Prdm9 speciation gene across diverse metazoan taxa.

The onset of prezygotic and postzygotic barriers to gene flow between populations is a hallmark of speciation. One of the earliest postzygotic isolating barriers to arise between incipient species is the sterility of the heterogametic sex in interspecies' hybrids. Four genes that underlie hybrid sterility have been identified in animals: Odysseus, JYalpha, and Overdrive in Drosophila and Prdm9 (Meisetz) in mice. Mouse Prdm9 encodes a protein with a KRAB motif, a histone methyltransferase domain and several zinc fingers. The difference of a single zinc finger distinguishes Prdm9 alleles that cause hybrid sterility from those that do not. We find that concerted evolution and positive selection have rapidly altered the number and sequence of Prdm9 zinc fingers across 13 rodent genomes. The patterns of positive selection in Prdm9 zinc fingers imply that rapid evolution has acted on the interface between the Prdm9 protein and the DNA sequences to which it binds. Similar patterns are apparent for Prdm9 zinc fingers for diverse metazoans, including primates. Indeed, allelic variation at the DNA-binding positions of human PRDM9 zinc fingers show significant association with decreased risk of infertility. Prdm9 thus plays a role in determining male sterility both between species (mouse) and within species (human). The recurrent episodes of positive selection acting on Prdm9 suggest that the DNA sequences to which it binds must also be evolving rapidly. Our findings do not identify the nature of the underlying DNA sequences, but argue against the proposed role of Prdm9 as an essential transcription factor in mouse meiosis. We propose a hypothetical model in which incompatibilities between Prdm9-binding specificity and satellite DNAs provide the molecular basis for Prdm9-mediated hybrid sterility. We suggest that Prdm9 should be investigated as a candidate gene in other instances of hybrid sterility in metazoans.